Both oligopeptides are the analogues of the hemoregulating pentapeptide isolated from natural source. The isolation of a low molecular mass factor from a matured granulocytes and from the suspension of rat medulla (bone-marrow) was first described by Rytomaa and Kiviniemi. This factor inhibited the proliferation of myelopoietic rat cells in in vitro tests (Cell Tissue Kinet., 1, 329 and 341 (1968)).
Granulocyte chalon, as it was named at the beginning, turned out to slow down the proliferation in the S-phase of the cell cycle and to inhibit it in the S.sub.1 -phase (Virchows Arch. Abt. B. Zellpath., 14, 293 (1973)) while it inhibits the same in the G.sub.1 -phase of the cell cycle (In Vitro, 4, 47 (1969)).
The first homogenously purified myelopoietic inhibiting factor was described in Cell Biol. Intereact. Rep., 4, 337 (1980). This factor belongs to the nucleopeptides regarding the structure thereof. The isolation, approximate structure analysis and synthesis thereof was first reported in Z. Naturforsch., 37.c, 1297 (1982).
In 1986 it was declared that the synthetic Xaa Glu Asp Cys Lys where Xaa is pyroglutamic acid pentapeptide exhibits the same biological effects as the substance isolated from natural source (Biological Regulation of Cell Proliferation, edited by Basega, Foa, Netcalf and Polli, Raven Press, New York, 1986, page 11). This pentapeptide is SEQ I.D. No. 5.
So far the hemoregulating peptide has been isolated from human granulocytes, rat medulla and calf spleen. The myelopoiesis inhibiting effect thereof upon mouse, rat and human cells is dose-dependent in a concentration of higher than 10.sup.-13 mole/l. It has almost no side-effect. It is not toxic to human and mouse bone marrow cells in a concentration range of 10.sup.-15 to 10.sup.-4 mole/l in a 7-day in vitro treatment and to two mouse species in a dose of 9.1 mg/animal or 1 mg/animal (Pharmac. Ther., 44, 353 (1989)).
On the basis of the former facts it is not surprising that the hemoregulating peptide is considered as a future drug which will protect the bone marrow from the damage caused by cytostatic agents and radiation used for the treatment of tumorous diseases; however its use in other pharmaceutical fields may also be promising.
None of the few analogues synthesized so far has been found to exhibit similar biological activity. Several data indicate that the difficulties arising in the structural elucidation of the pentapeptide, consisting of only three-functional amino acids in addition to the amino-terminal pyroglutamic acid, the problems of the different synthesis routes and the deviation of the biological activity of the samples are in close relation with the susceptibility to decomposition of the compound due to its structure.
In Cancer Res., 50, 328 (1990), the authors cell the attention to the oxidative dimerization of the compound carrying a mercapto group and to its inactivation in aqueous solution under repeated freezing/melting. Its susceptibility to dimerization is especially disadvantageous as the activity of the dimer is the opposite of that of the monomer (Exp. Hematol., 12, 7. (1984); Exp. Hematol., 16, 274 (1988)).
No explanation is given in the article for the lack of activity of certain synthetic samples and inactivation. In our opinion these phenomena can at least partially be attributed to the ring-closure, isomerization and epimerization of compounds containing aspartic acid (J. Chem. Soc. Chem. Commun., 1983, 505; Acta Chim. Hung., 124, 919 (1987); Peptides 1986, editor: Theodoropulos, de Gruyter, Berlin, 1987, 83; Coll. Czech. Chem. Commun., 54, 3360 (1989)).
The opposite, i.e. cell proliferation stimulating effect of the dimer molecule Xaa Glu Asp Cys Lys Xaa Glu Asp Cys Lys and the ineffectiveness of Xaa Glu Asp Met Lys where Xaa is pyroglutamic acid (SEQ I.D. Nos: 3 and 4 respectively) on cell proliferation indicate that the mercapto group of cysteine has a significant role in eliciting the biological activity. The necessity of the presence of the mercapto group is supported by the fact that the natural SEQ I.D. No. 5 pentapeptide can inhibit the proliferation only in mercapto ethanol of 0.1 to 0.3 mmole/l, while without mercapto ethanol it is ineffective.
Among others, it is surprising that the SEQ I.D. No: 1 pentapeptide containing an acetamidomethyl group and the SEQ I.D. No: 2 tetrapeptide shortened at the amino-terminus, exhibit a significant and selective inhibiting effect upon the proliferation of myeloid cells.
The effect upon cell proliferation can be measured by several methods. The tests may measure e.g. the incorporation of tritiated thymidine into DNA in short-term cell (rat thymus and bone marrow cell) cultures or determine the colony-forming ability of mouse bone marrow cells in semi-solid agar culture.